MR imaging of the human body can display both normal anatomy and a variety of organ pathologies, including tumors. For example, the liver, pancreas, spleen and gall bladder can be imaged by tomographic slices in various planes. The techniques used for MRI liver examination have included delineation by spinecho, inversion recovery, and saturation recovery pulse sequences, but definition of normal from abnormal has not been predictable. In hepatic MRI, specifically, contrast resolution of the hepatic images varies greatly depending on the data acquisition technique employed to obtain the image although tumors associated with the liver or spleen usually result in prolongation of both the longitudinal (T1) and the transverse (T2) relaxation times as compared with normal tissues. Earlier reports have emphasized the importance of using paramagnetic agents to increase the T1 differences between normal and pathologic tissues and considered coincidental T2 diminution an impediment.
Paramagnetic contrast agents as free metal ions, chelates, or insoluble metal compounds have been described for use in enhancing intrinsic contrast in MR imaging. Such paramagnetic metals include gadolinium, chromium, copper, manganese and iron. Because of possible toxicity, soluble chelates have been suggested for parenteral administration and insolubilized compounds for oral administration. Heretofore, however, the targeting of stable contrast agents to the liver and spleen has not been satisfactory.
Effective, safe reticuloendothelial system (RES) MRI contrast agent which can increase the sensitivity and differentiation of normal and pathologic tissue in the liver or spleen have been proposed in the art. However, existing modalities for other imaging procedures for the liver and spleen have approximately a 10-20% false-negative rate for detecting hepatic metastases, and a 40-50% false-negative rate for detecting lymphomatous involvement, necessitating laproscopic staging. Further, as pointed out above, tumor involvement of liver, spleen and other tissues has consistently been shown to increase T1 and T2 relaxation parameters to a variable and unpredictable degree, which also results in a high incidence of false-negative. RES agents are useful because liver replaced by tumor does not possess RES cells and therefore does not take up the contrast agent. Non-RES agents more randomly distribute between normal and pathologic tissue.
Hepatic disease conditions resulting in abnormally high levels of iron in the liver have been shown to produce laterations of tissue relaxation times as observed by MRI. See, for example, Doyle et al., AM J. Roentgenol (1982) 138: 193-200; Stark et al, Radiology (1983) 148: 743-751; and Runge, et al., Am. J. Roentgenol (1983) 141: 943-948. Observed decreases in T1 have been attributed either to paramagnetic enhancement of longitudinal relaxation, or to alterations of hydrated tissue proteins. Heretofore, the production of T2 diminution as seen in these disease states has not been produced with a potent, safe contrast agent. Soluble iron compounds have been tested as MRI contrast agents. Wesbey, et al Radiology (1983) 149: 175-180.
The foregoing discussion is taken largely from Widder U.S. Pat. No. 4,675,173 who proposed MRI examination employing encapsulated paramagnetic contrast agents. According to Widder the contrast agents preferably are ferromagnetic as well as paramagnetic. By utilizing microspheres within the size range of 1.5 to 8 microns, such as 3 to 5 microns the parenterally administered contrast agents reportedly are rapidly segregated by the reticuloendothelial system and concentrated in the liver and spleen. Effective segregation and concentration in these organs is said to occur in as short a time as 1 to 10 minutes, and only a small quantity of the microspheres reportedly needs to be administered for effective reduction of the T2 relaxation time of the subject's liver and/or spleen. Actually, by the early 1960's, the first stable magnetic fluid colloid had been described. Later research led to the development of a separations device based on magnetic density gradients in magnetic fluid columns. By 1979, magnetic particles coated with appropriate functional chemical grounds for affinity chromatography separations were reported.
For example, dextran/magnetite has been explored (see Hasegawa et al, U.S. Pat. No. 4,101,435; Molday, U.S. Pat. No. 4,454,773, and Schroder, U.S. Pat. No. 4,501,726). The complexes of dextran and iron oxide have had some success, but all have high molecular weight coatings (at least 500,000) which reportedly lead to adverse reactions in clinical trials. Apparently, such high molecular weight coatings dissociate, leaving the metal oxide free to aggregate.
Similarly, in 1985 Nycomed disclosed their efforts (e.g. PCT appln. WO85/02772) towards the development of particles for contrast agent applications. It was reported that it was best to fully enclose magnetic particles in a matrix which is "biocompatible", and that matrix materials included carbohydrates, polyamino acids (albumin) and certain synthetic polymers (acrylates, polystyrene, etc.).
Chan et al report preparing compounds known as "Ferrosomes". Invest. Radiol. 27(6), pp. 443-49 (1992). These are lipid-coated iron oxide particles and such particles were used as contrast agents. In a similar manner, Eli Lilly has reported on drug carrier formulations (U.S. Pat. No. 4,331,654) consisting of magnetically localizable, biodegradable, lipid microspheres.
The first commercial application of magnetic separations was described by Chagnon et al in U.S. Pat. No. 4,268,037. The Chagnon patent describes the use of amine terminated silane coupled magnetic particles for immunodiagnostic applications. The materials described in the Chagnon et al patent are now used commercially in medical diagnostic kits.
Magnetic separations have not been exclusively applied to in vitro applications. The use of magnetic separations for in vivo applications is becoming increasingly more accepted and important as a therapeutic and diagnostic tool. By the early 1980's published reports described the magnetic targeting and isolation of chemotherapeutic drugs into rat-tail sarcoma. Widder (U.S. Pat. Nos. 4,849,210; 4,247,406; and 4,230,685) described the use of magnetic albumin spheres for ultrasound contrast media and magnetic drug targeting. See also Widder U.S. Pat. Nos. 4,357,259; 4,345,588; and 5,179,955. And, Schroder (U.S. Pat. No. 4,501,726) reports a method of preparing magnetic starch beads for use in MRI imaging for the separation of T1/T2 relaxation signals. See also U.S. Pat. Nos. 4,770,183; 4,827,945 and 4,331,654.
Liposomes have received a great deal of attention over the past due to their ability to carry large amounts of therapeutic agents with decreased toxicity (Mayer, L. D., Bally, M. B., Loughrey, H., Masin, D., Cullins, P. R. (1990) Cancer Research 50, 575-579, Lopez-Berstein, G., Fidler, I. J., Liss (1989), New York pp 353-365 Juliano; A. L. Stamp D. (1978) "Pharmacokinetics of Liposomes-Encapsulated Antitumor Drugs," Biochem Pharmacal 28, 21-27), and recently as a protective delivery system of oligonucleotides (Thierry, A., Rahman, A. Dritschilo, A., "Liposomal Delivery As a New Approach to Transport Antisense Oligonucleotides" Gene Regulation: Biology of Antisense RNA and DNA, Raven Press N.Y. (1992). Lipid vesicle are formed around the drug of choice and the material is transported through the blood stream to an endothelial barrier. Crude targeting, done by size mediation, and chemical targeting done by using liposomes with antibodies on their surface, have been studied (Hughs, B., Kennel, S., Lee R., Huang, L., (1989) Monoclonal Antibody Targeting of Liposomes to Mouse Lung", In Vivo Cancer Research 49, 6214-6220 (1989) The liposome binds to the endothelial target, the drug is released from the liposome, transcytosed across the membrane and delivered to the tissue. This approach has suffered from the following three shortcomings: Liposomes have difficulty crossing the endothelium intact with the drug. The drug is therefore subject to entrapment in the basement membrane and lysosomal degradation after release from the liposome. Liposomes are not sheer stable, and the size discrimination targeting is inhibited by liposome instability in the bloodstream. Finally, attempts to target liposomes using antibodies have resulted in the only limited success (Papahakjopoulos, D., Gabizon, A., "Targeting of Liposomes to Tumor Cells" In Vivo Ann, N.Y., Acad. Sci. 507:64-74(87).
Several novel nanoparticle systems have been explored for a variety of purposes over the past several years. Microporous polymer beads that are less than 100 nm in diameter have been demonstrated to be effective in evading capture by the RES. Systems have been designed using these porous polymer vehicles loaded with a drug to slowly release nanoparticles and controlled dosages of drugs into the blood over a long period of time (Ruxandra, G., Minamitake, Y. Peachia, M., Trubetsky, V. Langer, "Biodegradable Long-Circulating Polymeric Nanospheres", Science Vol. 263, (1994). Iron oxide that has the ability to target hepatocytes has also been evaluated for MR imaging. (Josephson, L., Groman, E. Menz, E., Lewis, J., and Bengele, H. "A Functionalized Superparamagnetic Iron Oxide Colloid as a Receptor Directed MR Contrast Agent, Magn. Reson. Imag. 8,637-646, (1990); Reimer, P., Weissleder, R., Lee, A., Wittenberg, J., Brady, T., "Receptor Imaging: Application to MR Imaging of Liver Cancer," Radiology 177:729-734 (1990); Weissleder, R., Lee, A., Khous, B., Shen, T., Brady, T., "Antimyosin-l Labeled Monocrystalline Iron Oxide Allows Detection of Myocardial Infarct", Radiology 182,381-385 (1992).
The ideal drug delivery system is one that can transport a therapeutic agent, avoid capture by the RES, be targeted to a specific cell population, concentrate therapeutic levels of the drug at the site and be capable of releasing the drug to the targeted area in a controlled fashion. The clinical goal is to increase the therapeutic index of the drug by decreasing its systemic dosage while increasing the locally effective dosage by concentrating the agent at the site where it is needed. This strategy, designed to increase the desired effects and to decrease side effects of many drugs, may make it possible to utilize therapeutically beneficial molecules that currently can not be used because of the toxic effects that they cause when administered systematically.
In all of this previous work, the use of magnetic particles as contrast agents for magnetic resonance imaging (MRI) has been limited to the organs of the RES due to its rapid blood clearance. However, an advantage in enhanced separations, for example, could be achieved if the magnetic particles could be altered to avoid particular uptake by the RES. Solid particles have the advantages of having sheer resistant size, a controllable shape, carrying a predictable and stable surface charge and maintaining a uniform stable size distribution both in vitro and in vivo. Inorganic particle cores are of particular interest because of their biodegradability. Particles of sufficiently small diameter with hydrophilic surfaces that evade RES uptake have been synthesized and studied (Chagnon, M., Carter, M., Ferris, J., Gray, M., Hamilton, Tr., Rudd, E., "Preparation of Controlled Size Inorganic Particles For Use In Separations, As Magnetic Molecular Switches, As An Inorganic Lipsomes for Medical Applications," International Publication Number WO 93/26019 (1993).
Iron oxide particles with a significant decrease in RES recognition and an increase in blood circulation time would therefore be extremely beneficial. Reducing recognition by the RES would result in an increased circulation time for particulates which would permit development of an MRI agent for perfusion. By coupling specific targeting chemistries, the resulting material also could be used for imaging discrete areas of the body such as tumors and/or for targeting drug delivery.